JP2631867B2 - Method for producing (R) -2-hydroxy-4-phenyl-3-butenoic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial application field) The present invention relates to (R) -2-hydroxy-4-phenyl-3.
-To a method for producing butenoic acid. (R) -2-hydroxy-4-phenyl-3-butenoic acid is an important intermediate such as various pharmaceuticals, optically active physiologically active substances, and derivatives thereof.

(Prior Art and Problems to be Solved by the Invention) Conventionally, (R) -2-hydroxy-4-phenyl-3-
As a method for producing butenoic acid, a method is known in which a racemate of the acid is formed into a diastereomer with bornylamine and the resulting solution is optically resolved (Chem. Ber. 89, 671-677 (1956)), but the operation is complicated. It is difficult to say that it is industrially superior,
A simple and economical method was desired. Further, by utilizing the asymmetric reduction ability of microorganisms, 2-keto-4-phenyl-3-butenoic acid can be converted to (R) -2-hydroxy-4-phenyl-3-butene.
There is no known method for obtaining butenoic acid.

(Means for Solving the Problems) The present inventors used a simple method and asymmetric reduction by a microorganism as a method for obtaining (R) -2-hydroxy-4-phenyl-3-butenoic acid having high optical purity. Focus on the method,
As a result of searching for a microorganism suitable for this purpose, it was found that a microorganism belonging to the genus Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Sporololactobacillus achieves the object of the present invention, and completed the present invention. Things.

That is, the present invention relates to a lactobacillus, a leucono having the ability to asymmetrically reduce 2-keto-4-phenyl-3-butenoic acid to (R) -2-hydroxy-4-phenyl-3-butenoic acid. A microorganism selected from the group of microorganisms belonging to the genus Stock, Streptococcus, Pediococcus, and Sporolactobacillus or a processed product thereof is allowed to act on 2-keto-4-phenyl-3-butenoic acid to produce (R)- 2
A method for producing (R) -2-hydroxy-4-phenyl-3-butenoic acid, comprising collecting -hydroxy-4-phenyl-3-butenoic acid.

As the microorganism used in the present invention, Lactobacillus,
In microorganisms belonging to the genus Leuconostoc, Streptococcus, Pediococcus, and Sporolactobacillus, 2-keto-4-phenyl-3-butenoic acid does not differ from (R) -2-hydroxy-4-phenyl-3-butenoic acid. Any substance capable of simultaneously reducing can be used, and specifically, Lactobacillus lactis (Lactobacillus
lactis) AHU1059, Streptococcus faecalis IFO12964, Leuconostoc dextranicum I
FO3349, Bediococcus acidi Ractesh (Pedi
ococcus acidilactici) NRIC1102, Sporolactobacillus inulinus NR
IC1133 and the like can be mentioned. These mutants can also be used.

The medium used in the present invention is not particularly limited as long as the medium can grow the bacteria. For example, glucose, sugars such as sucrose, ethanol, alcohols such as glycerol, acetic acid, organic acids such as propionic acid, hydrocarbons such as paraffin, other carbon sources, or a mixture thereof, as a nitrogen source, ammonium sulfate, A nutrient source used for ordinary culture, such as an inorganic organic nitrogen-containing compound such as yeast extract and urea, an inorganic salt, a trace metal salt, and vitamins can be appropriately mixed and used. If necessary, a factor for promoting the growth of microorganisms or a compound for maintaining the pH of the medium can be added.

As the culture method, the medium pH is 3.0 to 9.5, and the culture temperature is 20 to
It is preferable to culture aerobically or anaerobically in the range of 45 ° C for 1 to 5 days.

As a method of the reduction reaction, (1) a method using a culture solution as it is, (2) a bacterial cell was separated by centrifugation or the like, and this was washed as it was or resuspended in a buffer solution, water or the like. A method in which 2-keto-4-phenyl-3-butenoic acid is added thereto and reacted (3) A crushed cell, treated with acetone, freeze-dried, or the like is used instead of viable cells. Method (4) A method in which these are immobilized on a carrier and used can be applied. In this reaction, the yield is often improved by adding a carbon source such as glucose or sucrose as an energy source.

2-keto-4-phenyl-3-butenoic acid as is
Alternatively, they may be dissolved in an organic solvent that does not affect the reaction, or dispersed in a surfactant or the like, and added all at once or dividedly from the beginning of the reaction. Further, 2-keto-4-phenyl-3-butenoic acid can be used as a sodium salt, a potassium salt, a calcium salt, an ammonium salt and the like.

The reaction is preferably carried out at a temperature in the range of 3 to 9 at a temperature of 10 to 60 ° C, and is carried out for 1 to 120 hours under stirring. The concentration of the substrate used is not particularly limited, but is preferably about 0.1 to 10%.

The 2-hydroxy-4-phenyl-3-butenoic acid produced by the reaction can be collected directly from the reaction solution or after acidification after cell separation, extraction with an organic solvent, and ordinary column chromatography, distillation and the like. It can be easily obtained by using a purification method.

(Examples) Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

The optical purity in Examples was determined by extracting the reaction product with an organic solvent, and then performing high-performance liquid chromatography using an optical resolution column (column: Daicel Chemical Industries, Chiral Pack WH (4.6 mm ID × 250 mm), moving layer: 0.5 mM CuSO). ₄ / acetonitrile = 4: 1, detection; 254 nm, flow rate 1.5 ml / min). (Retention time: (S) 21 minutes, (R) 24 minutes) The yield was determined by high performance liquid chromatography using a reversed phase column (column: Nucleosyl 10C18, (4.6 mm ID × 250 m
m) Mobile phase: 40 mM potassium phosphate, pH 3.0 / acetonitrile = 4: 1, detection; 254 nm, flow rate 1.0 ml / min).

Example 1 2% glucose, 1.0% yeast extract, 1.0% peptone,
Medium 10 consisting of 10 ppm MnSO ₄ and 1% calcium carbonate
0 ml was placed in a 500 ml Erlenmeyer flask, and after sterilization, each of the strains shown in Table 1 was inoculated with one platinum wire and cultivated by rotary shaking for 48 hours. After completion of the culture, the cells were separated by centrifugation and washed once with physiological saline to obtain viable cells. 50 ml of distilled water was placed in a 500 ml Erlenmeyer flask, the viable cells were suspended therein, and 5 g of sucrose was added. After shaking at 30 ° C for 10 minutes,
0.5 g of potassium keto-4-phenyl-3-butenoate
The mixture was added and allowed to undergo a rotary shaking reaction at 30 ° C. for 40 hours. After the reaction is completed, sulfuric acid is added to adjust the pH to 1 or less, and then ethyl acetate 100 ml
Extracted. After desolvating the extract, 2-hydroxy-4-produced using high performance fluid chromatography.
The quantification of phenyl-3-butenoic acid and the measurement of optical purity were performed.

 Table 1 shows the obtained results.

Example 2 A 5 L jar fermenter containing 2 L of the medium used in Example 1 was inoculated with Leuconostoc dextranicum IFO03349, and cultured at 30 ° C. with stirring at 100 rpm for 40 hours. After completion of the culture, the cells were collected by centrifugation and washed with 1 L of water. Thereafter, the cells were suspended in 500 ml of water, 5 g of potassium 2-keto-4-phenyl-3-butenoate, glucose
50 g and 5 g of calcium carbonate were added and reacted at 30 ° C. with stirring for 48 hours. After completion of the reaction, the mixture was adjusted to pH 1 with sulfuric acid, and extracted twice with an equal volume of ethyl acetate. After the ethyl acetate layer was dehydrated with anhydrous sodium sulfate, the solvent was removed under reduced pressure to obtain 4.0 g of crude crystals of 2-hydroxy-4-phenyl-3-butenoic acid. This was recrystallized from methanol to obtain 3.8 g of crystals of (R) -2-hydroxy-4-phenyl-3-butenoic acid. (Optical purity
(Effect of the Invention) (R) -2 by the asymmetric reduction method using the microorganism of the present invention.
The method for producing -hydroxy-4-phenyl-3-butenoic acid enables easy production of (R) -2-hydroxy-4-phenyl-3-butenoic acid having a high optical purity, and This is extremely advantageous as a production method.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12P 7/42 C12R 1:46)

Claims (1)

(57) [Claims] 1. Lactobacillus having the ability to asymmetrically reduce 2-keto-4-phenyl-3-butenoic acid to (R) -2-hydroxy-4-phenyl-3-butenoic acid. , Leuconosto
c), Streptococcus, Pediococcus, Sporolactobacillus (Spo
(R) -2-hydroxy- produced by reacting a microorganism selected from the group of microorganisms belonging to the genus rolactobacillus or a processed product thereof with 2-keto-4-phenyl-3-butenoic acid.
A method for producing (R) -2-hydroxy-4-phenyl-3-butenoic acid, comprising collecting 4-phenyl-3-butenoic acid.